Ceiling culture of mature human adipocytes: use in studies of adipocyte functions

Abstract

Adipocytes contain large lipid droplets in their cytoplasm. When cultured, they float on top of the medium, clump together, and do not gain equal and sufficient access to the medium. Morphological changes cannot be observed and the majority of adipocytes undergo cell lysis within 72 h of isolation. We have used a ceiling culture method for human mature adipocytes which uses their buoyant property to allow them to adhere to a floating glass surface, where they remain viable for several weeks. Using confocal immunofluorescence microscopy we showed the cellular expression and subcellular localization of leptin in ceiling-cultured adipocytes. The secretion of leptin was increased from ceiling cultures following tumour necrosis factor-treatment. Proliferation of mature human adipocytes in serum-containing medium was demonstrated by incorporation of bromodeoxyuridine, 2% of adipocytes showing positive incorporation after 4 h labelling. Proliferation was also evident from the budding of daughter cells. Apoptosis in the ceiling cultures was increased by 48 h serum deprivation (30-35 vs 10-15% in the control) and was assayed by propidium iodide staining and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labelling. Lipolysis, analysed by liquid scintillation counting, was increased by forskolin (10 µM for 90 min) and lipogenesis, shown by autoradiography, was stimulated by insulin (10 and 100 nM for 4 h). These findings indicate that ceiling-cultured adipocytes maintain adipocyte-specific functions and that ceiling culture, which overcomes the shortcomings of adipocyte suspension culture, can be used to study adipocyte cell biology.

Dedifferentiated fat cells: an alternative source of adult multipotent cells from the adipose tissues

“…PCR analysis showed that although DFAT cells had decreased level of LPL, leptin and glucose transporter 4 (GLUT4) compared with mature adipocytes, they still express key adipogenic markers including peroxisome proliferator-activated receptor gamma (PPARγ), CAAT/ enhancer-binding proteins (C/EBPα, C/EBPβ and C/EBPδ) and sterol regulatory element-binding protein-1c (SREBP1c) [14,[24][25]. In vitro, several recipes of adipogenic cultures were applied with different combination and dosage of induction chemicals such as dexamethasone, 3-isobutyl-1-methylxanthine and insulin transferring selenium X (ITS) [14][15]18]. Initial establishment of lipid droplets could found as early as 5-7 days of induction, while significant amount of lipid accumulation within cells could be observed after 2-3 weeks.…”

“…The adipocytes, other than the cell groups within the SVF portion, can be dedifferentiated into fibroblast-like cells named the dedifferentiated fat (DFAT) cells by ceiling culture strategy based on their buoyant property [13][14][15]. With this method and provided the fact that the mature adipocytes are the most abundant cell type in the adipose tissue, the DFAT cells which exhibit proliferation and multipotent capacity in vitro and in vivo have been regarded as an ideal source for adult postnatal pluripotent cells of much higher homogeneity than ASCs [14,[16][17][18][19].…”

Angiotensin II stimulates and atrial natriuretic peptide inhibits human visceral adipocyte growth

“…For more than 20 years it was clearly shown that in vitro-cultured mature unilocular adipocytes are able to become multilocular adipocytes and to undergo cell replication, 11 as it was confirmed later. 12 Because Ang II appears to have a trophic role in adipose tissue expansion, it may also contribute to the development of insulin resistance and type 2 diabetes. The improved insulin sensitivity and the reduced new-onset diabetes incidence in patients treated with angiotensin-converting enzyme inhibitors or AT1 receptor blockers 13,14 may be due to a 'brake' on the rate of VAT expansion.…”

Implantation of primary cultured adipocytes that secrete insulin modifies blood glucose levels in diabetic mice

“…Isolation of human fat tissue is being established as liposuction in plastic surgery and cosmetic surgery [15,16]. Human PAs can also be obtained by ceiling culture [17]. Our investigations may thus provide a basis for the treatment of patients by long-term supplementation of therapeutic protein without systemic immunosuppression.…”